Although substantial progress has been made in the prevention and treatment of cardiovascular disease and its major risk factors, it has been predicted that thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity worldwide well into the year 2020. Thrombosis remains the most prevalent cause of fatal diseases in developed countries. More than 80% of stroke cases are of thrombotic origin, and stroke is among the three leading causes of mortality and severe chronic disability in the US. Systemic anticoagulants like heparin prevent co-morbidity from deep vein thrombosis and reduce the progression of acute cerebral thrombosis. However, their bleeding side effects in the acute phase outweigh the benefits. An antithrombotic agent that can be administered to patients with severe acute thrombotic diseases, such as heart attack and stroke, without the risk of causing hemorrhage, would revolutionize the treatment of cardiovascular and cerebrovascular diseases. Protein engineering of thrombin has led to the development of the mutant W215A/E217A (WE) that is capable of sustained anticoagulant effects in non-human primates without bleeding complications. WE has compromised activity toward fibrinogen and PARI, but retains activity toward the anticoagulant protein C. Activation of protein C by WE on the endothelium is more effective in producing an anticoagulant effect than systemic administration of activated protein C. In addition to exploiting the protein C anticoagulant pathway, WE is capable of producing a profound antiplatelet effect by engaging the Gplb receptor and preventing its interaction with von Willebrand factor, which is a property not present in either activated protein C or heparin. Filing of an IND for the mutant WE is on track for August 2012. The remarkable success of WE in pre-clinical studies and its extraordinary promise as an effective anticoagulant/antithrombotic may be compromised in humans by its residual activity toward fibrinogen and PARI. The goal of this research project is to produce a new generation of thrombin mutants that have completely lost their activity toward fibrinogen and PARI, but retain activity toward protein C in the presence of the cofactor thrombomodulin.